Miller K R, Parks J S
Department of Comparative Medicine, Bowman Gray School of Medicine, Wake Forest University, Winston-Salem, NC 27157, USA.
J Lipid Res. 1997 Jun;38(6):1094-102.
Interfacial binding affinities and capacities of lecithin:cholesterol acyltransferase (LCAT) and apolipoprotein A-I (apoA-I) for surfaces of different phosphatidylcholine (PC) composition, cholesterol content, and apolipoprotein content were measured with a vesicle model system. Native polyacrylamide gel electrophoresis was used to separate free protein from vesicle-bound protein. ApoA-I was isolated from human plasma and radiolabeled with iodine, whereas radiolabeled LCAT was purified from the media of Chinese hamster ovary cells that were transfected with human LCAT cDNA and incubated in the presence of [35S] cysteine and methionine. Bound and free radiolabeled LCAT and apoA-I were quantified by phosphorimage analysis. ApoA-I binding was not influenced by cholesterol content (14 mole%) but was influenced by the PC fatty acyl composition of the vesicle. PC species containing long chain, polyunsaturated fatty acids (PUFA) in the sn-2 position resulted in increased binding affinity (Kd = 75-177 nM) but reduced capacity (0.1-0.3 apoA-I/ 1000 PC) in comparison to sn-1 palmitoyl, sn-2 oleoyl PC (POPC, 750 nM and 1.4 apoA-I/1000 PC). LCAT binding affinity to POPC (2190 nM) was stronger in the presence of cholesterol (530 nM), and LCAT binding capacity was reduced (2.63 and 0.6 molecules LCAT/1000 PC, respectively). In comparison to POPC, LCAT binding affinity to sn-1 palmitoyl, sn-2 arachidonyl PC was stronger (611 nM) and binding capacity was reduced (0.7 LCAT/1000 PC). LCAT binding affinity and capacity to sn-1 palmitoyl, sn-2 eicosapentaneoyl PC (2041 nM, and 2.5 LCAT/1000 PC) were similar to those observed for POPC. We conclude that vesicle surface PC fatty acyl composition and cholesterol content significantly influence LCAT and apoA-I interfacial binding and therefore may alter LCAT enzymatic activity.
利用囊泡模型系统测定了卵磷脂胆固醇酰基转移酶（LCAT）和载脂蛋白A-I（apoA-I）对不同磷脂酰胆碱（PC）组成、胆固醇含量和载脂蛋白含量的表面的界面结合亲和力和容量。采用天然聚丙烯酰胺凝胶电泳将游离蛋白与结合在囊泡上的蛋白分离。从人血浆中分离出apoA-I并用碘进行放射性标记，而放射性标记的LCAT则从转染了人LCAT cDNA并在[35S]半胱氨酸和甲硫氨酸存在下孵育的中国仓鼠卵巢细胞培养基中纯化得到。通过磷图像分析对结合和游离的放射性标记的LCAT和apoA-I进行定量。apoA-I的结合不受胆固醇含量（14摩尔%）的影响，但受囊泡PC脂肪酸酰基组成的影响。与sn-1棕榈酰基、sn-2油酰基PC（POPC，750 nM和1.4 apoA-I/1000 PC）相比，在sn-2位置含有长链多不饱和脂肪酸（PUFA）的PC种类导致结合亲和力增加（Kd = 75 - 177 nM）但容量降低（0.1 - 0.3 apoA-I/1000 PC）。在胆固醇存在的情况下，LCAT对POPC的结合亲和力（2190 nM）更强（530 nM），并且LCAT的结合容量降低（分别为2.63和0.6个LCAT分子/1000 PC）。与POPC相比，LCAT对sn-1棕榈酰基、sn-2花生四烯酰基PC的结合亲和力更强（611 nM）且结合容量降低（0.7 LCAT/1000 PC）。LCAT对sn-1棕榈酰基、sn-2二十碳五烯酰基PC的结合亲和力和容量（2041 nM和2.5 LCAT/1000 PC）与对POPC观察到的相似。我们得出结论，囊泡表面PC脂肪酸酰基组成和胆固醇含量显著影响LCAT和apoA-I的界面结合，因此可能改变LCAT的酶活性。
